Coupled AbioticBiotic Mineralization of 2,4,6-Trinitrotoluene (TNT)

Thomas F. Hess* and Paul S. Schrader

ABSTRACT

Fentons reaction is the catalyzed decomposition of

hydrogen peroxide by transition metals, resulting in the
generation of hydroxyl radicals (Haber and Weiss, 1934).
The standard Fenton reaction proceeds by adding dilute
hydrogen peroxide to a degassed solution of iron(II),
resulting in nearly stoichiometric generation of hydroxyl
radicals. Most environmental applications of Fenton
chemistry involve reaction modifications, including use
of higher concentrations of hydrogen peroxide, phosphate-buffered medium, heterogeneous catalysts, or iron.
These conditions, although not as stoichiometrically efficient as the standard Fentons reaction, are often necessary to treat sorbed contaminants in soils and ground
water (Tyre et al., 1991).
Hydroxyl radical has high reactivity with many environmental contaminants at or near diffusion-controlled
rates (109 M1 s1 ). The degradation of xenobiotic
chemicals by hydroxyl radical is typically due to either
hydroxylation or hydrogen atom abstraction. Some biorefractory compounds such as perchloroethylene, hexachlorocyclopentadiene, and hexachlorobenzene have
been effectively destroyed by hydroxyl radicals within
minutes (Leung et al., 1992; Sato et al., 1993; Watts et
al., 1994). The use of Fentons reagent (29.4 mM H2O2
and 40 mM Fe2 ) for the destruction of TNT in aqueous
solutions (0.31 mM) resulted in complete degradation
within 8 h and 40% mineralization within 24 h (Li et
al., 1997a).
Anaerobic and aerobic biodegradation processes
have been investigated for the destruction of TNT.
While aerobic TNT biodegradation has been demonstrated, problems such as accumulation of metabolic
intermediates (Vorbeck et al., 1994, Ramos et al., 1995),
inhibitory intermediate compound formation (Michels
and Gottschalk, 1995), and low mineralization (Fernando et al., 1990) limit process effectiveness. Additionally, reductive microbial transformation of TNT is the
thermodynamically favorable pathway due to the aromatic ring stabilization and electron-withdrawing effect
of the nitro groups (Bruhn et al., 1987). The consensus
of recent research is that anaerobic biological processes
hold the most promise for stand-alone bioremediation
of TNT (Funk et al., 1993; Preuss and Rieger, 1995,
Crawford, 1995; Regan and Crawford, 1994; Lewis et
al., 1997). The research has indicated, however, that
substantial mineralization of TNT may not be achieved
although the parent compound can be entirely transformed to intermediary metabolites (Crawford, 1995).
Combined technologies for the destruction of hazardous wastes have received research attention (Carberry
and Benzing, 1991; Koyama et al., 1994; Scott and Ollis,
1995; Ravikumar and Gurol, 1991). In these studies, the
authors investigated sequential processes, using abiotic

Munitions wastes such as TNT are widespread contaminants in

soils and ground waters. We investigated a coupled abioticbiotic
treatment scheme for remediation of aqueous solutions of TNT. Mineralization of aqueous TNT (0.22 mM ) was initially optimized with
minimum reactant use (Fe3 and H2O2 ) in light-assisted and dark,
modified Fenton reactions at acidic and neutral pH. Complete TNT
degradation occurred under all reaction conditions within 24 h. Using
the optimum reactant concentrations, coupled abioticbiotic reactions
showed an increase in TNT mineralization, from 47 to 80%, after
biomass addition to the acidic, dark Fenton-like reaction. Comparable
increases of TNT mineralization were observed under neutral pH
with similar reaction conditions. In light-assisted Fenton-like reactions
at neutral pH, no increase in cumulative TNT mineralization (66%)
was seen in coupled abioticbiotic reactions. Abiotic photo-Fentonlike reactions alone, at acidic pH, produced complete TNT mineralization and required no biotic assistance. While light-enhanced Fenton
reactions alone can provide high levels of TNT mineralization, the
dark abioticbiotic reaction scheme has perhaps a wider use due to
a similar extent of TNT mineralization in the absence of light, leading
to possible applications in soil slurry and in situ processes in the subsurface.

aste generated during the past manufacture of

2,4,6-trinitrotoluene (TNT) and decommissioning
of military ordnance has contributed to widespread environmental contamination at many current and former
Department of Defense (DOD) facilities located
throughout the United States (Urbanski, 1964; Spalding
and Fulton, 1988). More than 1200 explosives-contaminated DOD sites have been identified with almost 90%
of these sites containing TNT-contaminated ground water (Schmelling and Gray, 1995). Because TNT is acutely
toxic to humans and animals even in low concentrations
(Watts, 1998), mutagenic in the Ames test (Won et al.,
1976), and listed as a priority pollutant by the USEPA
(Schuster and Gratzfeld-Huesgen, 1993), remediation
of TNT-contaminated soils and ground waters is legally mandated.
The numerous technologies investigated for remediation of waters contaminated with TNT and related compounds principally have been based on chemical or biological processes. Studies of chemical treatment have
focused on advanced oxidative processes (AOPs), while
those related to biological transformation have used
either bacterial or fungal systems under aerobic or anaerobic conditions. The AOPs previously applied to the
treatment of TNT include ozone-catalyzed decomposition of TNT (Lang et al., 1998), TiO2mediated photocatalysis (Schmelling et al., 1996; Schmelling and Gray,
1993, 1995), and Fenton chemistry (Li et al., 1997a,b).
Center for Hazardous Waste Remediation and Research, Univ. of
Idaho, Moscow, ID 83844-0904. Received 14 May 2001. *Corresponding author (tfhess@uidaho.edu).

Abbreviations: DNP, 2,4-dinitrophenol; NTA, nitrilotriaceticacid;

TNT, 2,4,6-trinitrotoluene; WAS, waste-activated sludge.

Published in J. Environ. Qual. 31:736744 (2002).

736

HESS & SCHRADER: COUPLED ABIOTICBIOTIC MINERALIZATION OF TNT

reactions as a pretreatment step for a separate, followon biological reaction. Such technologies were developed to overcome the biorecalcitrance of a particular
compound inherent with stand-alone biological processes. Our own research into sequential, coupled processes has indicated that TiO2mediated photocatalysis
followed by biological degradation (Hess et al., 1998)
and coexistent abiotic and biotic transformations (Buyuksonmez et al., 1998, 1999; Howsakeng et al., 2002)
can be used to treat biorefractory compounds.
This research was designed to test the efficacy of
coupled abioticbiotic reactions for a high extent of
TNT destruction in simple, aqueous matrices, thus
avoiding complications of soil solutions, yet provide a
basis for future work in soil. A modified Fentons system
(Fe3 catalyst) was used in the abiotic reaction and two
different, uncharacterized aerobic biomasses were tested for their potential to degrade any Fenton-degradation products. The specific objectives of this research
were fourfold: (i) explore the efficacy of the chemical
treatment under a variety of environmental conditions,
(ii) optimize the chemical mineralization of TNT under
these conditions, (iii) explore the use of an aerobic microbial biomass as a subsequent treatment, and (iv) determine the kinetics of each treatment.
MATERIALS AND METHODS
Chemical Reagents
Iron(III) sulfate pentahydrate (97%) and nitrilotriaceticacid (NTA, 99%) were purchased from Aldrich Chemical
Company (Milwaukee, WI). Reagent-grade H2O2 (30% v/v)
was obtained from J.T. Baker (Phillipsburg, NJ). The 2,4,6trinitrotoluene (99%) was purchased from Chem Service
(West Chester, PA). Ecolite () scintillation cocktail was purchased from ICN Biomedicals (Costa Mesa, CA). Uniformly
ring-labeled 2,4,6-trinitrotoluene with a specific activity of 2.18
MBq mM1 (99%) was synthesized by Dr. Stefan Goszczyn-

737

ski of the Environmental Biotechnology Institute, University

of Idaho (Moscow, ID). All other chemicals used in the project
were of the highest available purity. Double-deionized water
(18 M-cm) was used for preparation of all chemical solutions.

Experimental Design
Several sets of experiments were conducted for this research: abiotic, optimization experiments to determine maximum TNT mineralization occurring at minimum reactant concentrations; combined abioticbiotic kinetic experiments
(conducted at optimum reactant concentrations determined
above) to determine any increase in TNT mineralization due
to biotic reactions; abiotic degradation experiments conducted
to determine extent of TNT degradation using optimum reactant concentrations; and abiotic experiments to determine
the effect of metal chelate concentrations on overall TNT
mineralization (Fig. 1). Modified Fenton reactions were used
for all abiotic TNT mineralization and degradation studies.
Optimization experiments were originally conceived as twolevel, rotatable, central composite designs (Table 1) but later
converted to factorial designs when early data (dark-Fenton
reactions, pH 3) did not show rotatability (Table 2) (Cochran
and Cox, 1992). The two-level designs (Tables 1 and 2) included Fe3 and H2O2 concentrations as experimental variables with TNT mineralization as the response. The factorial
experimental design was used to quantify the effects of the
individual experimental variables, iron and hydrogen peroxide, on the response. This was done by analyzing the response
when testing one experimental variable over a range of concentrations while leaving the other constant. The same was
then done for the other variable. Optimality was determined
graphically based on the maximum response achieved using
the lowest reactant concentrations.

Abiotic TNT Mineralization Experiments

Abiotic TNT mineralization was measured in both optimization and kinetic experiments by capture and quantification
of 14CO2 produced during the modified Fenton reactions. All

Fig. 1. Diagram of experimental setup showing incremental use of results (arrows) between various stages of experimentation.

738

J. ENVIRON. QUAL., VOL. 31, MAYJUNE 2002

Table 1. Experimental designs and results of 14C-labeled experiments for investigation of the effects of iron and hydrogen
peroxide on the mineralization of aqueous TNT (0.22 mM ) in
dark-Fenton reactions at pH 3. Optimum iron and hydrogen
peroxide concentrations that produced maximum TNT mineralization are in italic type.
Fe3

H2O2
mM

2.93
2.93
17.07
17.07
0.00
20
10
10
10
10
10
10
10
0.01
0.05
0.1
0.5
1
2
3
5
0.01
0.05
0.1
0.5
1
2
3
5
1
2
3
5
0.01
0.05
0.1
0.5
1
2
3
5

TNT mineralization
%

Central composite design

43
251
43
251
147
147
0
294
147
147
147
147
147
Factorial design
15
15
15
15
15
15
15
15
29
29
29
29
29
29
29
29
44
44
44
44
74
74
74
74
74
74
74
74

30.73
37.79
27.61
33.54
0
31.73
0
34.09
31.58
32.65
33.02
33.08
33.84
0.32
0.55
1.52
31.03
27.45
29.85
27.53
24.47
0.36
0.98
1.87
29.75
31.60
34.56
28.47
27.51
37.33
32.87
32.44
29.19
0.52
1.58
2.31
24.70
42.68
32.35
33.97
31.33

experiments were conducted with 30-mL solutions of TNT

(0.22 mM ) using mixtures of nonlabeled TNT and enough 14C
uniformly labelled TNT to provide approximately 1.67 103
Bq per flask. Mineralization reactions were conducted in 500mL biometer flasks each sealed with a rubber stopper and
containing a glass cup (holding base solution) suspended in
the atmosphere of the flask and a piece of glass tubing extending from the atmosphere of the flask through the stopper.
Attached to the outside end of the glass tubing was an expandable bladder used to hold the gasses evolved from the reaction
and allow free exchange with the flask atmosphere. The glass
cup contained 1 mL of a 0.1 M NaOH solution used to capture
CO2 from the flask atmosphere. For optimization experiments,
the 1-mL NaOH sample was collected, as well as two successive 1-mL H2O-rinsates, at the end of the experiment. For
kinetic experiments, the sample and rinsates were collected
at timed intervals during the course of the experiment. The
samples were added directly to 15 mL of Ecolite () liquid
scintillation cocktail and analyzed by scintillation counting as
described below to quantify the amount of 14CO2 generated
from the reaction. Knowing the original quantity of 14C-TNT
in solution, we determined the extent (percent) of TNT miner-

Table 2. Experimental design and results of 14C-labeled experiments for investigation of the effects of iron and hydrogen
peroxide on the mineralization of aqueous TNT (0.22 mM ) in
dark-Fenton reactions at pH 7 and light-Fenton reactions at
pH 3 and pH 7. Experiments at pH 7 used NTA to Fe molar
ratios of 10:1 to chelate iron. Optimum iron hydrogen peroxide
concentrations that produced maximum TNT mineralization
are in italic type.
Factorial design
Fe

H2O2

TNT mineralization
Light, pH 3

Dark, pH 7

Light, pH 7

1.29
5.84
81.16
89.26
84.21
80.94
75.29
63.19
59.28
4.40
6.56
94.67
95.00
92.05
88.11
82.83
79.35
71.57
5.31
9.56
94.05
99.35
97.63
93.44
90.75
82.88
81.88
5.31
7.98
80.34
99.15
97.93
96.67
91.31
76.86
78.28
2.40
5.99
53.74
91.78
84.42
88.67
87.84
85.49
79.57

%
3.72
6.05
17.55
16.72
10.41
7.66
4.01
2.48
1.76
1.83
4.97
22.81
17.78
16.70
10.90
5.23
2.74
0.38
2.83
3.03
24.88
20.49
21.21
19.13
12.99
5.48
1.82
7.76
14.49
23.95
16.25
14.47
12.21
8.96
7.27
2.25
5.24
9.69
20.77
15.16
11.15
9.64
6.01
4.55
2.25

9.57
19.08
33.44
23.11
12.75
7.53
3.46
1.13
0.00
15.93
24.58
29.44
36.34
25.17
16.60
7.92
3.03
0.87
25.76
28.40
28.01
22.07
32.69
25.98
16.11
5.84
1.76
18.28
23.88
27.72
16.63
14.55
11.91
10.48
7.95
2.32
12.05
15.91
23.12
16.25
9.73
9.47
7.29
4.24
2.02

mM
0.05
0.1
0.5
1
2
3
5
10
20
0.05
0.1
0.5
1
2
3
5
10
20
0.05
0.1
0.5
1
2
3
5
10
20
0.05
0.1
0.5
1
2
3
5
10
20
0.05
0.1
0.5
1
2
3
5
10
20

15
15
15
15
15
15
15
15
15
29
29
29
29
29
29
29
29
29
74
74
74
74
74
74
74
74
74
147
147
147
147
147
147
147
147
147
294
294
294
294
294
294
294
294
294

alization. A 1-mL sample of the aqueous portion of the reaction was collected at the end of the experiment and analyzed
similarly to determine a mass balance on 14C.
Procedurally, the mineralization experiments were set up
by initially adding Fe3 to the TNT solution and the Fentonlike reaction was then begun by adding H2O2 to the TNTFe3
solution. Hydrogen peroxide and Fe3 concentrations varied
between 15 and 294 mM (1%) and 0.05 and 20 mM, respectively, for optimization experiments (Tables 1 and 2) and were
constant for kinetic experiments, based on results found during
optimization (values listed in italic type in Tables 1 and 2).
The iron used in neutral pH reactions was chelated with nitrilotriaceticacid (NTA) at either equimolar concentrations (1:1)
or ten times more NTA than iron (10:1), depending on the
experiment. The pH of the NTAFe3 solution was adjusted
to 7.0 using NaOH. All experiments were monitored for pH
using a meter and probe, and calibrated prior to each use with
standard buffer solutions (Accument Basic; Fisher Scientific,

HESS & SCHRADER: COUPLED ABIOTICBIOTIC MINERALIZATION OF TNT

Pittsburgh, PA). Experiments performed in the dark (darkFenton) were conducted after covering the reaction vessels
in aluminum foil. Those experiments run in the light (lightFenton) were put under a light box containing six, 24-inch
full-spectrum (380750 nm, peak intensity at 610 nm) 20-watt
light bulbs, 16-inches above the flasks, giving a light intensity
of 54.2 cd m2.

Biotic Reactions
Kinetic experiments that received biotic treatment subsequent to the Fenton reaction were first brought to neutral pH
by the addition of 1 mL of M9 salts (Provence and Curtiss,
1994). The addition of the salts resulted in a final concentration
of Na2HPO4 (42.3 mM ), KH2PO4 (22 mM ), NaCl (8.5 mM ),
and NH4Cl (18.7 mM ). Two uncharacterized biomasses from
aerobic, bench-scale sequencing batch reactors (SBRs), described previously (Hess et al., 1993), were used in separate
experiments. The first SBR was seeded with waste-activated
sludge (WAS) from the Pullman, Washington Wastewater
Treatment Facility, fed daily a synthetic waste (Kennedy et
al., 1990) with an organic carbon content of approximately
130 mg L1, and maintained at an average total suspended
solids (TSS) concentration of 2800 mg L1. The WAS was
added to the biometer flasks at 24 h after the initiation of the
abiotic, modified Fenton reaction (after solution neutralization) at an average concentration of either 467 or 93 mg L1,
depending on the experiment, and allowed to react for an
additional 6 d with base samples, containing 14CO2, taken at
regular intervals. The second SBR was seeded with a consortium of 2,4-dinitrophenol (DNP)degrading bacteria, fed daily
with a DNPglucose waste (Hess et al., 1990) with an organic
carbon content of approximately 45 mg L1, and maintained
at an average TSS concentration of 2000 mg L1. This DNP
biomass was added to biometer flasks similar to the WAS
biomass at concentrations of 333 and 67 mg L1, depending
on the experiment.

Liquid Scintillation Analysis

All samples from mineralization studies were counted with
a liquid scintillation analyzer (Tri-Carb Model 2100TR; Packard Bioscience, Meriden, CT) using a 14C protocol. Counts
per minute were converted to disintegrations per minute by
using an efficiency plot for known 14C quench standards and
appropriate blanks to eliminate background chemiluminescence.

TNT Degradation Sample Quenching

and Preparation
For analyses of each TNT degradation experiment, a 2-mL
sample of the modified Fenton reaction mixture was combined
with 100 L of sodium bicarbonate (approximately 0.9 M ) in
a test tube in order to stop the reaction (Glaze and Kang,
1988). A 1-mL aliquot of the quenched material was prepared
for high performance liquid chromatography (HPLC) analysis
by filtering through a 0.2-m nylon filter and placed into 1.5mL amber vials with Teflon-lined septa.

Chemical Analyses
The TNT concentrations were determined using HPLC
(Model 1090, Series II; Hewlett Packard, Palo Alto, CA)
equipped with a security guard column containing a C18 (ODS
octadecyl) filter connected to a C18 reverse-phase column
(250 mm 2.0 mm 5 m; Phenomenex, Torrance, CA). A
binary solvent, gradient elution methodology was used and

739

consisted of (i) acetonitrile and (ii) 0.5 mM lithium phosphate

buffer, pH 4.0 0.1, at a flow rate of 0.22 mL min1. Initial
conditions were 5% acetonitrile (0 to 3 min), to 51% acetonitrile (3 to 21 min, held 12 min), to 70% acetonitrile (21 to 29
min, held 4 min), with a return to initial setup conditions by
32 min. A 10-L injection of each sample collected was analyzed with the temperature of the HPLC column held constant
at 40C. The HPLC was equipped with a diode array UV/
Visible light detector (DAD) monitoring A230 with continuous
scanning of the absorption spectrum of each peak from 190
to 600 nm. Compounds detected were identified by a comparison of their retention times and UV/Visible light spectra with
those of authentic standards.
Identification of nitrate and oxalate ions was determined
by ion chromatography using conductivity detection (Dionex
Corporation, Sunnyvale, CA). A binary solvent, isocratic elution methodology was used for separation and consisted of
89.5% double distilled deionized water and 11.5% 100 mM
NaOH solution. Total run time for each sample was 9 min.
An IonPac AG-11 (Dionex) guard column connected to an
IonPac AS11 column with an IonPac ATC-1 filter for carbonate removal were used for all separations. Anions detected
were identified and quantified based on comparison with known
reference standards.

RESULTS AND DISCUSSION

Two major groups of experiments were conducted in
this project: (i) optimization experiments, using abiotic
Fenton reactions, designed to find minimum reactant
concentrations resulting in maximum TNT mineralization; and (ii) kinetic experiments, using abiotic Fenton reactions (conducted at reactant concentrations as
found in optimization experiments) coupled with follow-on biotic reactions, to determine the increase in
TNT mineralization due to biological treatment. Both
groups of experiments were conducted with four different reaction conditions: light-Fenton and dark-Fenton
reactions at pH 3 and light-Fenton and dark-Fenton
reactions at pH 7. Two additional minor groups of experiments were conducted, based on optimization results: (i) determination of the effects of iron chelate
concentrations on TNT mineralization extent and (ii)
overall TNT degradation kinetics. All experiments used
0.22 mM aqueous TNT solutions with varying concentrations of Fe3 and H2O2 in the modified Fenton reactions. The biotic portion of kinetic experiments used
varying concentrations of two uncharacterized biomasses, previously described.

Abiotic TNT Mineralization Optimization

Optimal concentrations of reactants, Fe3 and H2O2,
in aqueous, modified Fenton reactions were determined
from the results of experiments in factorial designs (Tables 1 and 2) for the four reaction conditions described
above. Initial experiments were based on rotatable, central composite designs (dark-Fenton reactions at pH 3)
but, upon failing to show rotatability, were converted
to factorial designs more appropriate for investigating
the resulting maximal responses (Chochran and Cox,
1992). Iron(III) and hydrogen peroxide were tested over
concentration ranges of 0.05 to 20 mM and 15 to 294
mM, respectively. Optimal reactant concentrations, the

740

J. ENVIRON. QUAL., VOL. 31, MAYJUNE 2002

Table 3. Percent recovery of 14C in optimization (Tables 1 and 2) and kinetic (Fig. 35) experiments related to aqueous TNT (0.22 mM )
mineralization. Data are means standard error (P 0.05).
14

Kinetic experiments (n 3)

Optimization
experiments

Abiotic biotic (WAS)

Abiotic
Dark, pH 3
Dark, pH 7
Light, pH 3
Light, pH 7
Biotic control

92.8
91.7
89.3
90.0

5.5
2.0
1.7
1.7

(n
(n
(n
(n

Abiotic

C recovery

41)
45)
45)
45)

89.4
100.25
103.4
100.0

0.9
0.8
4.6
0.4

93 mg

L 1

%
88.7 1.4
97.4 0.3
93.8 1.7

Abiotic biotic (DNP)

L 1

67 mg L1

300 mg L1

81.7 6.8
90.1 1.4

90.5 4.8
92.9 1.8

75.7 1.2
85.3 6.7

93.9 1.7
99.5 0.7

94.3 2.1

93.9 0.9
97.8 2.1

467 mg

Waste-activated sludge.
2,4-Dinitrophenol biomass.

lowest concentrations of reactants producing the highest

extent of TNT mineralization (shown in italic type in
Tables 1 and 2) were determined graphically (not
shown) for each experiment. These reactant concentrations were used for all subsequent experimentation. Experimental error for the data (Tables 1 and 2) was approximately 0.8% mineralization, based on the standard
deviation of the mean of center point replicates of the
central composite experiment (Table 1). Carbon balances, based on recovery of 14C (Table 3), were done
for all optimization data (Tables 1 and 2) and averaged
approximately 90% recovery for each reaction condition.
Several phenomena were observed upon examination
of the results. In all of the optimization experiments,
TNT mineralization extent declined with reactant concentrations above optimal (Table 1), presumably due
to competitive reactions between the reactive oxygen
radical species and excess Fe3 (De Laat and Gallard,
1999). Greater TNT mineralization extent in light-Fenton reactions (Table 2) than in dark-Fenton reactions
(Table 1), under similar conditions, was attributed to
photo-assisted Fenton oxidation, mechanistically described by others (Sun and Pignatello, 1993; Li et al.,
1997a). Overall TNT mineralization extent in dark-Fenton reactions was considerably less under neutral pH
conditions than under acidic conditions. This was attributed to either or both (i) a lowered efficiency of hydroxyl
radical formation at neutral pH or (ii) a higher reactivity
of hydroxyl radical with the ironchelate complex than
with TNT. The chelating agent (NTA) was required to
prevent formation of iron oxide precipitates at neutral
pH, thus removing the soluble iron catalyst from solution and limiting the Fenton-like reaction.

to iron ratio resulted in the greatest TNT mineralization

extent of 66%, whereas the same reaction with a 10:1
ratio produced 44% TNT mineralization (data not
shown). Similarly, with dark-Fenton reactions, the lower
molar ratio of NTA produced 45% TNT mineralization
versus 28% for the reaction with a 10:1 molar ratio of
NTA to Fe (data not shown).

TNT Degradation
Degradation of TNT has been a common benchmark
for success of a treatment process (Schmelling and Gray,
1993; Schmelling et al., 1996) and a good complement
to measuring the mineralization of TNT. By using both
measurements, one may determine how much of the
TNT parent molecule remains as well as the fraction
mineralized. Degradation experiments were performed
for the four light- and dark-Fenton reactions using the
optimal reactant and chelate concentrations defined
previously. Two additional light- and dark-Fenton reactions, at neutral pH, were also conducted with optimal
reactant concentrations and excess chelate to iron concentrations (10:1). It was observed (Fig. 2) that 100% of

Effect of Chelate Concentration

As mentioned above, the efficiency of Fenton reactions with TNT at neutral pH was lowered, presumably
due to the use of a chelate. We therefore explored the
effects of different chelate (NTA) concentrations on
TNT mineralization in light- and dark-Fenton reactions
at neutral pH. Optimal concentrations of iron(III) and
hydrogen peroxide (Tables 1 and 2) were used in these
experiments. A 1:1 molar ratio of chelate to iron was
the lowest concentration of NTA that maintained iron
in solution. The light-Fenton reaction with a 1:1 NTA

Fig. 2. Degradation of aqueous TNT (0.22 mM ) in dark- or lightFenton reactions: dark, pH 3 (); light, pH 3 (); light, pH 7 (Fe3
to nitrilotriaceticacid [NTA] ratio 1:1) (); dark, pH 7 (Fe3 to
NTA ratio 1:1) (); light, pH 7 (Fe3 to NTA ratio 1:10) ();
dark, pH 7 (Fe3 to NTA ratio 1:10) (). See text for description
of dark- and light-Fenton reactions. Error bars on symbols indicate
standard error of means (n 3, P 0.05); where absent, bars fall
within symbols.

HESS & SCHRADER: COUPLED ABIOTICBIOTIC MINERALIZATION OF TNT

the TNT was transformed under each reaction condition

within 24 h and, in most cases, within 6 h. The effect of
lowering the chelate concentration was seen in these
degradation experiments. The dark-Fenton reaction at
neutral pH, and containing a 10:1 chelate to iron ratio,
required nearly 24 h for complete TNT degradation
as compared with 10 h for an experiment with similar
reaction conditions, but with a 1:1 chelate to iron ratio.
Photo-Fenton effects were also observed (Fig. 2). More
time was required for complete TNT degradation in all
dark-Fenton reactions (624 h) compared with lightFenton reactions (26 h).

Coupled AbioticBiotic TNT

Mineralization Kinetics
The kinetics of a treatment process must be known
prior to assessing its overall viability, cost, and usefulness in an engineered system (Grady et al., 1999).
We therefore conducted several sets of experiments to
determine (i) the kinetics of the abiotic, modified Fenton reaction and (ii) the kinetics of a combined, sequential abioticbiotic reaction scheme using modified
Fenton reactions followed by biological treatment. All
Fenton reactions were investigated using the optimal
reactant concentrations previously determined. The abi-

741

otic reaction was run for 24 h at the pH tested with

subsequent addition of biomass after solution neutralization.
Capture and analysis of 14CO2 resulting from biological or chemical transformation of radiolabeled compounds is widely regarded as incontrovertible evidence
of compound mineralization (Bartha and Pramer, 1965;
Code of Federal Regulations, 1996). Using modified
biometer flasks, described above, we conducted kinetic
studies on the abiotic mineralization of TNT in darkFenton reactions, at both pH 3 and 7. The cumulative
TNT mineralization achieved in each reaction condition,
47 and 45% respectively, was approximately 85% complete within 24 h (Fig. 3). The extent of TNT mineralization in dark-Fenton reactions at neutral pH (45%) was
greater than that seen in the optimization experiments
(25%) due to the lowered NTA to Fe ratio (1:1) found
during chelate optimization studies. Kinetic studies using dark-Fenton reactions with a NTA to Fe ratio of
10:1 (similar to the initial optimization studies), at neutral pH, yielded an ultimate TNT mineralization extent
of 28% (data not shown).
In an effort to increase TNT mineralization using a
coupled abioticbiotic scheme, experiments were conducted with the addition of biotic treatment after the
abiotic, modified Fenton treatments previously investi-

Fig. 3. Mineralization of aqueous TNT (0.22 mM ) in dark-Fenton reactions comparing abiotic with coupled abioticbiotic treatments at high
and low biomass (waste-activated sludge [WAS] or 2,4-dinitrophenol [DNP] biomass) concentrations: (A ) pH 3, WAS biomass; (B ) pH 3,
DNP biomass; (C ) pH 7, WAS biomass; (D ) pH 7, DNP biomass. Abiotic reaction (); high biomass concentration, either 467 mg WAS
L1 or 333 mg DNP L1 (); low biomass concentration, either 93 mg WAS L1 or 67 mg DNP L1 (). Abiotic, pH 7, reactions conducted
with Fe3 to nitrilotriaceticacid (NTA) ratio 1:1. Biotic reactions for (A ) and (B ) were conducted after solution neutralization. Error bars
on symbols indicate standard error of means (n 3, P 0.05); where absent, bars fall within symbols.

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J. ENVIRON. QUAL., VOL. 31, MAYJUNE 2002

gated. Two different biomasses were tested, a waste-activated sludge (WAS) biomass and a 2,4-dinitrophenol
(DNP)degrading biomass, both cultivated in benchscale sequencing batch reactors (Hess et al., 1993). Experiments were conducted with the four reaction conditions based on pH, acidic and neutral, and presence or
absence of light during the abiotic Fenton reactions.
The timing of biomass addition to the abiotic TNT reaction was critical and several time intervals after abiotic
reaction initiation (6, 10, and 14 h) were initially tested.
In all tests, TNT mineralization increased with the addition of biomass (data not shown). However, presumably
due to abiotic reaction quenching by the biotic, cellular
constituents (catalase and peroxidase), the overall coupled abioticbiotic TNT mineralization extent increased
with increasing time interval prior to biomass addition,
indicating improper timing of the addition. We therefore conducted further experiments with biomass addition 24 h after abiotic reaction initiation (Fig. 3). Overall
TNT mineralization increased from 47%, in the darkFenton reaction, pH 3, to 75 and 80% in the coupled
abioticbiotic system with WAS and DNP biomasses,
respectively (Fig. 3A,B). In reactions conducted at neutral pH, an increase in TNT mineralization from 45% in
the dark-Fenton reaction to 78% in both of the coupled
abioticbiotic systems (WAS or DNP biomasses) was
also seen (Fig. 3C,D). Direct TNT mineralization due
to biological action was negligible (less than 2%) based
on results from biotic controls (Fig. 4). Analysis of 14C
for all kinetic experiments indicated adequate carbon
balances (Table 3) with most experiments achieving at
least 90% recovery of radioactivity. Lower recovery percentages (90%) in some experiments were attributed
to experimental error.
The mechanism of increased TNT mineralization in
the coupled abioticbiotic system over that of the abiotic
reaction alone was probably due to biotic assimilation
of the abiotic TNT transformation products. As recently
reported by others (Li et al., 1997a), dicarboxylic acids,

Fig. 4. Biotic mineralization of aqueous TNT (0.22 mM ) with two

different biomasses, 467 mg waste-activated sludge (WAS) L1
() and 333 mg 2,4-dinitrophenol (DNP) L1 (). Error bars on
symbols indicate standard error of means (n 3, P 0.05); where
absent, bars fall within symbols.

principally oxalic acid, and nitrate are the primary end

products of dark-Fenton reactions with TNT. We also
found accumulation of oxalate and nitrate in solution
after abiotic reactions in approximate 1.4 and 3.1 molar
ratios to TNT, respectively (data not shown). In such
reaction conditions (low pH, dark reaction), oxalate is
essentially unreactive with hydroxyl radical (KOH
4.7 107 mol s1; Buxton et al., 1988) and would persist
in solution. Oxalate is readily usable by many aerobic
organisms, entering their metabolic pathways after breakdown via oxalate decarboxylase (Tanner and Bornemann, 2000; Hokama et al., 2000). In our abioticbiotic
experiments, there was no statistical difference (P
0.05) between ultimate TNT mineralization extent (approximately 78 3%) for WAS or DNP biomasses, at
both acidic and neutral pH and low biomass concentrations (Fig. 3A,B and Fig. 3C,D, respectively), indicating
that both consortia of organisms probably used a common enzyme and/or pathway to degrade the byproducts
of Fenton-mediated transformation of TNT. Analysis
of the data in two experiments (Fig. 3B,C), however,
showed statistical differences between mineralization
kinetics in the biotic reaction for low and high concentrations of similar biomasses. In both experiments, the
low biomass concentrations (67 or 93 mg L1 as TSS)
produced a higher extent of TNT mineralization in a
shorter length of time than did the higher concentrations
of biomass (333 or 467 mg L1 as TSS). This phenomenon may have been due to quenching, by the biomass, of
the Fenton reaction, only approximately 85% complete
(Fig. 3), thereby slowing the biotic reaction and lowering
the overall TNT mineralization extent. Such solution
interactions probably also resulted in coexistent abiotic
and biotic reactions, recently shown to exist in combined
Fentonbiological reactions degrading perchloroethylene and its transformation products (Buyuksonmez et
al., 1999) and perchloroethylene and oxalate (Howsawkeng et al., 2002).
Results of kinetics studies on TNT mineralization in
light-Fenton reactions were quite different from those
in dark-Fenton reactions. Abiotic, modified Fenton reactions at pH 3, using optimal reactant concentrations
(Table 2) previously found, resulted in near-complete
mineralization of TNT (95%) within 48 h (data not
shown). The high extent of TNT mineralization, in
photo-assisted reactions, was similar to that found by
Li et al. (1997a), with the exception of our lower reactant
concentrations. Because of the essentially complete
mineralization of TNT at acidic pH when photo-assisted,
abiotic Fenton reactions were used, no follow-on biotic
reactions were necessary. However, at neutral pH, the
abiotic light-Fenton reaction (using optimal reactant
concentrations from Table 2 and a 1:1 ratio of NTA to
Fe3 ) resulted in only approximately 65% TNT mineralization (Fig. 5), presumably due to lowered efficiency
of hydroxyl radical formation as compared with acidic
pH. Surprisingly, when biomass was added to the abiotic
reaction mixture after 24 h of reaction (at approximately
90% of reaction completion), no significant increase in
TNT mineralization occurred. As seen in Fig. 5, two
different concentrations of WAS biomass and DNP bio-

HESS & SCHRADER: COUPLED ABIOTICBIOTIC MINERALIZATION OF TNT

743

Fig. 5. Mineralization of aqueous TNT (0.22 mM ) in light-Fenton reactions at pH 7 comparing abiotic with coupled abioticbiotic treatments
at high and low biomass (waste-activated sludge [WAS] or 2,4-dinitrophenol [DNP]) concentrations: (A ) WAS biomass; (B ) DNP biomass.
Abiotic reaction (); high biomass concentration, either 467 mg WAS L1 or 333 mg DNP L1 (); low biomass concentration, either 93
mg WAS L1 or 67 mg DNP L1 (). Abiotic reactions conducted with Fe3 to nitrilotriaceticacid (NTA) ratio 1:1. Error bars on symbols
indicate standard error of means (n 3, P 0.05); where absent, bars fall within symbols.

mass, respectively, failed to appreciably alter the abiotic

TNT mineralization kinetics.
The lack of biotic activity after abiotic light-Fenton
reactions was probably due to photo Fentoninduced
destruction of oxalate (or other Fenton transformation
products of TNT), the only microbially assimilable carbon in solution. Other researchers have found high a
extent of oxalate mineralization in photo-Fenton systems (Zuo and Hoigne, 1992; Li et al., 1997a). Such
phenomena are reportedly due to photodissociation of
the chelate in solution by ligand-to-metal charge transfer excitation followed by reaction of the photoreduced
iron with peroxide in the Fenton reaction (Sun and Pignatello, 1993).

CONCLUSIONS
Abiotic reactions were optimized for maximum TNT
mineralization using minimum reactants, Fe3 and H2O2,
under acidic pH and neutral pH conditions in modified
light- and dark-Fenton reactions. We demonstrated that
100% degradation could be obtained under each reaction condition, but the observed mineralization varied
widely. At acidic pH, light-Fenton reactions yielded
near complete mineralization of TNT (99%) versus
43% in dark-Fenton reactions. At neutral pH, light- and
dark-Fenton reactions produced a TNT mineralization
extent of 36 and 25%, respectively. Upon reducing the
chelate to iron molar ratio (from 10:1 to 1:1) in solution,
TNT mineralization was increased to 66% and 45% in
the light- and dark-Fenton reactions, respectively.
Addition of biomass to the abiotic reaction products
increased overall TNT mineralization extent at both
acidic and neutral pH in the modified dark-Fenton reactions. Using this coupled reaction process, abiotic (darkFenton reaction) TNT mineralization at pH 3 was increased from 47 to 75 or 80% using WAS biomass or
DNP biomass, respectively. Under neutral pH conditions (dark-Fenton reaction), TNT mineralization was
increased from 45 to 78% by both biomasses. The increase in TNT mineralization was probably due to the
abiotic formation of organic acids as intermediate prod-

ucts that were subsequently transformed by microorganisms.

Kinetics of the coupled abioticbiotic reactions were
comparatively rapid. The abiotic reaction required 24 h
while the addition of the biomass required an additional
4 d including the 10-h lag between the time of addition
of the biomass and an increase in the mineralization
of TNT. These kinetics are competitive with chemical
treatments and much faster than most stand-alone biological treatments.
Application of the coupled abioticbiotic process may
have the most utility when using the dark-Fenton reaction. In aqueous, reactor-based processes, a high extent
of TNT mineralization is achievable, comparable with
photo-Fenton reactions, with no additional power input
or concern for light penetration. A wider use of the
process, however, may be for the remediation of soils
contaminated with TNT, in both slurry reactor and in
situ treatments, where photo-Fenton processes are not
applicable. We are currently investigating the use of
these coupled processes for remediation of contaminated soils.
ACKNOWLEDGMENTS
Appreciation is expressed to NSF EPSCoR Cooperative
Agreement OSR-9350539 for funding assistance on this project.

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